Constitutive activation of pro-survival kinases has become a promising target of small molecules with an increasing interest in developing multi-targeted agents. The mechanisms underlying the responsiveness to most agents targeting cancer specific survival pathways are still poorly understood but critical for their clinical application. In this study, we found that sunitinib, a small molecule inhibitor of multiple tyrosine kinases including VEGFRs and PDGFRs induces apoptosis and inhibits cell growth in colon cancer cells in cell culture and xenograft models via the BH3-only protein PUMA. Sunitinib treatment induced PUMA transcription via the AKT-FoxO3a axis. PUMA, BH3 mimetics, or 5-Flurourical sensitized colon cancer cells to sunitinib-induced apoptosis. Furthermore, PUMA was induced by sunitinib treatment in xenograft tumors, and deficiency in PUMA significantly suppressed the anti-tumor effects of sunitinib. Our study suggests that PUMA-mediated apoptosis is important for the therapeutic responses to sunitinib, and activation of the mitochondrial pathway by BH3 mimetics or PUMA manipulation may be useful for improving the antitumor activity of sunitinib. Modulation of PUMA and selective Bcl-2 family members might be potential biomarkers for predicting sunitinib responses.